The Journal of Neuroscience, April 1, 2001, 21(7):2256–2267 Multiple Distinct Signal Pathways, Including an Autocrine Neurotrophic Mechanism, Contribute to the Survival-Promoting Effect of Depolarization on Spiral Ganglion Neurons In Vitro Marlan R. Hansen, Xiang-Ming Zha, Jinwoong Bok, and Steven H. Green Departments of Biological Sciences and Otolaryngology, University of Iowa, Iowa City, Iowa 52242 We have shown previously that BDNF, neurotrophin-3 (NT-3), nor do inhibitors of neurotrophin-dependent survival affect sur- chlorphenylthio-cAMP (cpt-cAMP) (a permeant cAMP analog), vival attributable to cpt-cAMP. However, Rp-cAMPS does par- and membrane depolarization promote spiral ganglion neuron tially inhibit depolarization-dependent survival, an inhibition (SGN) survival in vitro in an additive manner, depolarization that is additive with that by Trk-IgGs, PD98059, or LY294002. having the greatest efficacy. Expression of both BDNF and of Moreover, Rp-cAMPS prevents depolarization-dependent sur- NT-3 is detectable in cultured SGNs after plating in either vival of PC12 cells maintained in subthreshold levels of NGF. depolarizing or nondepolarizing medium. These neurotrophins Inhibition of Ca2ϩ/calmodulin-dependent protein kinases promote survival by an autocrine mechanism; TrkB-IgG or (CaMKs) with KN-62 reduces SGN survival independently of TrkC-IgG, which block neurotrophin binding to, respectively, Rp-cAMPS, Trk-IgGs, and LY294002 and additively with them. TrkB and TrkC, partially inhibit the trophic effect of depolariza- Combined inhibition of Trk, cAMP, and CaMK signaling pre- tion. The mitogen-activated protein kinase kinase inhibitor vents depolarization-dependent survival. Thus, survival of PD98059 and the phosphatidylinositol-3-OH kinase inhibitor SGNs under depolarizing conditions involves additivity among LY294002 both abolish trophic support by neurotrophins but a depolarization-independent autocrine pathway, a cAMP- only partially inhibit support by depolarization. Inhibition by dependent pathway, and a CaMK-dependent pathway. these compounds is not additive with inhibition by Trk-IgGs. Key words: membrane depolarization; spiral ganglion neuron; The cAMP antagonist Rp-adenosine-3Ј,5Ј-cyclic-phosphoro- cell survival; neurotrophic factor; BDNF; NT-3; cAMP; MAP thioate (Rp-cAMPS) abolishes survival attributable to cpt- kinase; ERK; phosphatidylinositol-3-OH kinase; signal trans- cAMP but has no effect on that attributable to neurotrophins, duction; autocrine mechanism Neuronal survival is supported by neurotrophic factors, such as ade of membrane electrical activity in vivo (Wright, 1981; Furber the neurotrophins, and by membrane depolarization. Substantial et al., 1987; Meriney et al., 1987; Maderdrut et al., 1988; Rubel et progress has been made recently in understanding the intracellu- al., 1990; Catsicas et al., 1992; Galli-Resta et al., 1993) and in vitro lar signals recruited by neurotrophic factors to prevent cell death (Lipton, 1986; Ruitjer et al., 1991) reduces neuronal survival. (Segal and Greenberg, 1996). Neurotrophins signal through the Depolarization permits neuronal survival in vitro in the absence Trk family of receptor protein-tyrosine kinases to activate at least of added neurotrophic factors (Scott and Fisher, 1970; Bennett two intracellular signal pathways implicated in promotion of and White, 1979; Chalazonitis and Fischbach, 1980; Wakade et neuronal survival: the Ras–MAP kinase (ERK) pathway (Xia et al., 1983; Gallo et al., 1987). ϩ ϩ al., 1995; Yan and Greene, 1998; Bonni et al., 1999) and the Ca2 influx through L-type Ca 2 channels appears to be a phosphatidylinositol-3-OH kinase (PI3K)–protein kinase B necessary first step (Gallo et al., 1987; Collins and Lile, 1989; (PKB, Akt) pathway (Yao and Cooper, 1995; D’Mello et al., Koike et al., 1989; Franklin et al., 1995; Hegarty et al., 1997) for ϩ 1997; Dudek et al., 1997; Miller et al., 1997). promotion of survival by depolarization, implicating Ca 2 as a ϩ Depolarization is an important trophic stimulus accounting, at second messenger for survival signaling. Ca2 /calmodulin- ϩ least in part, for neurotrophic support by presynaptic cells; block- dependent protein kinases (CaMKs) are activated by Ca2 (Hanson and Schulman, 1992) and thus could mediate Received Aug. 30, 1999; revised Nov. 28, 2000; accepted Dec. 7, 2000. depolarization-dependent survival, as shown by Hack et al. This work was supported by National Institutes of Health Grant DC02961 (1993). Also, cAMP is a survival signal for neurons (Rydel and (S.H.G.), an American Otological Society Research grant (S.H.G.), a grant from the Deafness Research Foundation (S.H.G.), and a grant from the Academy of Otolar- Greene, 1988; Kaiser and Lipton, 1990; D’Mello et al., 1993; Galli yngology (M.R.H.). M.R.H. was supported by National Institutes of Health Training et al., 1995; Meyer-Franke et al., 1995; Hegarty et al., 1997; ϩ Grant DC00040. These studies made use of facilities and services provided by the Hanson et al., 1998), and Ca 2 elevates intracellular cAMP University of Iowa Diabetes and Endocrinology Research Core, funded by National 2ϩ Institutes of Health Grant DK25295. We thank Becky Kueter for technical assis- levels via Ca /calmodulin-dependent adenylyl cyclase (Cooper tance, Dr. Stephen Heard for assistance with statistics, and members of the Green et al., 1995). Thus, a cAMP-dependent pathway might also me- laboratory for comments on this manuscript. We also thank Dr. Alan Saltiel diate survival-promoting effects of depolarization, as seems to be (Parke-Davis, Ann Arbor, MI) for providing PD98059 and Genentech for providing the neurotrophins, antibodies to neurotrophins, and Trk-IgG immunoadhesins used the case in retinal ganglion cells (Meyer-Franke et al., 1995). in these studies. Depolarization may promote survival by stimulating an autocrine Correspondence should be addressed to Steven H. Green, Department of Bio- logical Sciences, University of Iowa, 138 Biology Building, Iowa City, IA 52242- neurotrophic mechanism: synthesis and release of neurotrophic 1324. E-mail: [email protected]. factors by the neurons themselves (Ghosh et al., 1994). Neurotro- Copyright © 2001 Society for Neuroscience 0270-6474/01/212256-12$15.00/0 phins promote survival via PI3K or ERK pathways (Xia et al., Hansen et al. • Depolarization Trophic Signaling Mechanisms J. Neurosci., April 1, 2001, 21(7):2256–2267 2257 1995; Yao and Cooper, 1995; D’Mello et al., 1997; Dudek et al., Technologies, Gaithersburg, MD) was added to 10% to inhibit enzymatic 1997; Miller et al., 1997; Bonni et al., 1999; Mazzoni et al., 1999), activity, followed by three washes in serum-free DMEM and one wash in culture medium (see below). The ganglia were mechanically dissociated and depolarization can also activate these pathways (Rosen et al., using two fire-polished reduced-orifice glass Pasteur pipettes, the second 1994; Lev et al., 1995; Miller et al., 1997; Vaillant et al., 1999), considerably more narrow than the first. The ganglia were gently tritu- raising the possibility that one or both directly mediate promotion rated ϳ15 times with each pipette and diluted with culture medium (8–10 of survival by depolarization. Such a role for PI3K–PKB is ganglia/2 ml). supported by some (Miller et al., 1997; Vaillant et al., 1999) but Equal volumes of dissociated spiral ganglion cell suspension were plated in 96 well tissue culture plates (Corning, Corning, NY) that had not all (D’Mello et al., 1997) studies. been treated with polyornithine (0.1 mg/ml in 10 mM Na borate buffer) Studies of the auditory system highlight the significance of for 1 hr at room temperature, followed by laminin (20 ␮g/ml; Life afferent input in trophic support of neurons and the important Technologies) overnight at 4°C. Cells were grown in 100 ␮l of culture media at 37°C in a 6.5% CO2 incubator. Trophic agents and inhibitors role of electrical activity therein. Spiral ganglion neurons (SGNs) ϩ were added 2–3 hr after plating. In all chronic elevated [K ]o depolar- die after deafferentation because of loss of hair cells (Spoendlin, ϩ ϩ ization experiments, Na was replaced by equimolar K to maintain 1975; Webster and Webster, 1981; Koitchev et al., 1982; Bichler et osmolarity. al., 1983). Electrical stimulation via an electrode implanted in the Neuronal counts were performed after 48 hr in culture as described cochlea promotes survival of deafferented SGNs (Wong-Riley et previously (Hegarty et al., 1997). Briefly, the cells were fixed with fresh al., 1981; Lousteau, 1987; Hartshorn et al., 1991; Leake et al., 4% paraformaldehyde for 20 min at room temperature. The neurons 1991, 1992; Lustig et al., 1994), supporting a role for membrane were identified by immunocytochemistry: labeling with anti-neuron- specific enolase (NSE) antibody (Zymed, South San Francisco, CA), electrical activity in SGN survival. followed sequentially by a horse-radish peroxidase (HRP)-conjugated SGNs express TrkB and TrkC (Mou et al., 1997) and are goat anti-rabbit secondary antibody and visualization with 3-amino-9- supported in vitro by BDNF and by neurotrophin-3 (NT-3) (Avila ethylcarbazole. All neurons in each well were counted. Criteria for et al., 1993; Pirvola et al., 1994; Vazquez et al., 1994; Zheng et al., neuronal viability were NSE immunoreactivity and a nucleus that was visible and not pyknotic. Each condition was done in triplicate and 1995; Hegarty et al., 1997), as well as by a permeant cAMP analog repeated on at least three different occasions. (Hegarty et al.,